| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
University Department of Surgery, University of Edinburgh, Western General Hospital (E.S.G., M.B.R., F.K.H.), Edinburgh, Scotland EH4 2XU; and the Department of Discovery Biology Central Research Division, Pfizer Ltd. (S.B., A.N.), Kent, United Kingdom CT13 9NJ
Address all correspondence and requests for reprints to: Dr. E. S. Grant, University Department of Surgery, Western General Hospital, Crewe Road South, Edinburgh, Scotland EH4 2XU. E-mail: esg{at}srv0.med.ed.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IR3 (50 µg/mL) reduces the rate of stromal cell proliferation by
approximately 60–80% even in the presence of stimulatory
concentrations of IGFs. Camptothecin-induced apoptosis is inhibited by
the addition of IGF-I and -II (500 ng/mL). IR3 suppresses these
survival signals and itself induces cell death in the prostatic stroma.
The data suggest that IGF-IR is a pivotal molecule in prostatic stromal
cell maintenance, and that specific antagonism may offer a novel means
of controlling the fibromuscular expansion characteristic of benign
prostatic hyperplasia. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-subunits and two transmembrane ß-subunits
containing a cytoplasmic tyrosine kinase domain (1). IGF-IR gene
expression is a feature common to a diverse array of tissues, coupling
the ligands IGF-I, IGF-II, and insulin (binding affinities: IGF-I
> IGF-II > insulin) to intracellular differentiation,
metabolism, and, perhaps most crucially, mitogenesis (1, 2, 3). Indeed,
confirmation of the pivotal role played by the IGF-IR in the cell cycle
is evidenced by the complete abrogation of all requirements for
exogenous growth factors in BALB/c3T3 cells overexpressing the receptor
(4). Furthermore, it would appear that even when the type II IGF
(IGF-IIR) receptor is present in abundance, only the IGF-IR relays
IGF-I and -II proliferation signals (5, 6). Paralleled by this
growth-promoting function, the IGF-IR also has the potential for
modulating apoptosis. Apoptosis is decreased in cell lines that
overexpress the IGF-IR (7). In addition, apoptosis is induced by the
use of antisense oligonucleotides to the IGF receptor and by the
expression of a dominant negative mutant IGF-IR (8, 9). Both the
mitogenic and antiapoptotic functions of the IGF-IR are reliant upon
the tyrosine kinase activity of the ß-subunit (10, 11). Benign prostatic hyperplasia (BPH) describes a benign neoplasm characterized primarily by hyperplasia of the fibromuscular stroma (12, 13). Stromal cell cultures derived from BPH secrete detectable levels of IGF-II, but not IGF-I (14). In addition, it is apparent that these cells display elevated expression not only of IGF-II but also of IGF-IR compared with stromal cell cultures derived from normal prostate tissue (14, 15). This altered pattern of expression is believed to have its basis in lowered expression of the Wilm’s tumor gene WT-1, whose protein product is a known transcriptional regulator of the IGF-II and IGF-IR genes.
To establish the precise function of the IGF-IR within the fibromuscular stroma of the hyperplastic prostate, we examined the influence of IGF-IR activation and deactivation on BPH-derived stromal cell proliferation and apoptosis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Receptor grade human recombinant IGF-I was purchased from
Peninsula Laboratories Europe (St. Helens, UK). The levels of IGF-I and
IGF-II within culture-conditioned medium were determined using the DSL
Active IGF enzyme-linked immunosorbent assay (ELISA) systems
(Diagnostic Systems Laboratories, Webster, TX). The IGF antisera show
no cross-reactivity with IGF isoforms, insulin, or GH. The IR3
anti-IGF-IR antibody was purchased from Cambridge Bioscience
(Cambridge, UK). Clone IR3 binds an epitope within the -subunit
adjacent to the ligand-binding site and blocks IGF-I binding to its
receptor without affecting either the insulin or IGF-II receptors
(16).
Establishment of prostatic stromal cultures
Primary cultures of prostatic stroma were established using BPH tissue obtained from transurethral resections of the prostate (17). The benign status of the tissues was confirmed by histopathological examination. The cells were routinely maintained in RPMI 1640 (Life Technologies, Paisley, Scotland) supplemented with 10% FBS. Cultures at passages 0–4 were used in all analyses. The ligand binding studies and cell growth and apoptosis assays presented were performed on cultures derived from the same patient (BST1). The cultures were characterized using microscopy and immunohistochemistry with an antihuman smooth muscle myosin antibody and were found to contain a heterogeneous cell population composed of both fibroblasts and smooth muscle. Such characteristics were previously described by Kassen et al. (18).
IGF ELISA
Cells were grown in 12 mL serum-free medium (SFM/ts) supplemented with transferrin (10 µg/mL) and selenium (2 nmol/L) at 37 C for 72 h. Cell counts were performed at the end of the incubation period. After centrifugation at 1000 x g for 10 min to remove suspended cells, the medium was lyophilized and subsequently resuspended in 250 µl PBS. Fifty microliters of medium were taken for assay. Binding protein-associated IGF was released by incubation with a solution of ethanolic HCl before ELISA according to the manufacturer’s protocol. The concentrations of IGF are quoted in femtomoles per mL/106 cells after 72 h and represent the mean of three independent assays ± SE.
Preparation of total ribonucleic acid (RNA)
Total cellular RNA was extracted using the acid-guanidium-phenol-chloroform method (19).
RT-PCR amplification
Single stranded complementary DNA (cDNA) was synthesized from 5 µg total RNA using a commercial reverse transcription kit (Promega, Madison, WI) and the manufacturer’s protocol. Twenty percent of the cDNA was removed for PCR.
The PCR reactions were performed in a 1 x reaction buffer [Promega; 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.8), 1.5 mmol/L MgCl2, and 0.1% Triton X-100] containing 0.125 mmol/L of each deoxyribonucleotide triphosphate, 0.5 g of each primer, and 1 U Taq polymerase (Promega). Human IGF-IR cDNA sequences were detected using published primer sequences (20). Human IGF-IIR cDNA sequences were detected using intron-spanning primers derived from published sequences (21): sense, 5'-GTG ATG AAT ATG ACA ACC ACT G-3'; and antisense, 5'-CTT CTG ATG TCA AGA GAC AAT G-3'. Control PCR reactions were carried out using primers specific to the gene for the housekeeping protein hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (17). All samples were subjected to 35 cycles of PCR reaction. The IGF-IR PCR cycle was comprised of denaturation at 94 C for 1 min, annealing at 60 C for 1 min, and primer extension at 72 C for 2 min. The IGF-IIR PCR cycle comprised of denaturation at 94 C for 1 min, annealing at 58 C for 1 min, and primer extension at 72 C for 2 min. The PCR products were electrophoresed on 1.2% ethidium bromide gels and visualized under UV illumination. No RT and no DNA controls accompanied all PCR reactions (data not shown). Product authenticity was confirmed by restriction analysis (data not shown).
[125I]IGF-I binding assay
Stromal cells were preincubated in RPMI 1640–0.1% BSA
supplemented with transferrin (10 µg/mL) and selenium (2 nmol/L) for
20 min before the addition of 125I-labeled IGF-I or IGF-II
(Amersham, Little Chalfont, UK) to 0.1 nmol/L plus unlabeled IGFs or
insulin (Sigma-Aldrich, Poole, UK) in the concentration range 0.7
nmol/L to 0.5 µmol/L. After a 1-h incubation at 22 C, the cells were
washed three times in ice-cold PBS and subsequently solubilized in a
solution of 1% (vol/vol) Triton X-100. Cell-bound radioactivity was
assessed in duplicate by 
-counting. The apparent dissociation
constant (Kd) and the maximal binding capacity
(Bmax) were calculated by Scatchard analysis (22).
Cell proliferation
Stromal cells, seeded into 96-well plates at a density of 2000
cells/well, were incubated initially in their normal growth medium for
24 h at 37 C and then in SFM/ts supplemented with IGFs at 0.5–500
ng/mL. Cell growth kinetics were followed over 96 h. The influence
of IGF type I immunoneutralization on the IGF growth axis was assessed
by the addition of the IR3 IGF-IR monoclonal antibody (0.1–50.0
µg/mL), with or without IGF-I, for 48 h. Cell proliferation was
measured spectrophotometrically using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
(23). Each data point represents the mean OD540nm ±
SE.
DNA fragmentation ELISA
Photometric cell death detection ELISA (Boehringer Mannheim,
Lewes, UK) was performed to quantitate the apoptotic index by detecting
the histone-associated DNA fragments (mono- and oligonucleosomes)
generated by the apoptotic cells. The assay is based upon the
quantitative sandwich enzyme immunoassay principle, using monoclonal
antibodies raised against DNA and histones, respectively, for specific
determination of nucleosomes in the cytoplasmic fraction of cell
lysates. Cells were plated into 24-well culture plates (1 x
105 cells/well) in normal growth medium, grown for 24
h, then subsequently washed once with SFM/ts and treated with various
concentrations of the topoisomerase I inhibitor camptothecin (0.1, 1.0,
and 5.0 µg/mL; Sigma-Aldrich) with or without 500 ng/mL IGF-I in
SFM/ts for 24 h. The effects of IGF-IR immunoneutralization on
stromal cell death was ascertained using IR3 at 50.0 µg/mL. Cells
floating in the conditioned medium were pelleted and lysed along with
the remaining adherent cells, and the ELISA was performed according to
the manufacturer’s protocol. Each data point represents the mean
OD415nm-OD490nm of two independent tests.
Statistical analysis
Statistical significance was determined using Student’s t test for comparison of two means and the Bonferroni method (24) for comparison of a number of means against a control mean.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As noted previously (14), IGF-II, but not IGF-I, was detectable in
the conditioned medium of benign stromal cell cultures (Fig. 1
). The assay employed estimates an
IGF-II concentration of between 125–175 ng/mL·106 cells
in stromal cell-conditioned medium after 72 h. In contrast, the
levels of IGF-I measurable from the same cultures were at or below the
lower detection limits of the assay.
|
Analysis of total RNA isolated from three separate cell cultures
using an IGF-IR-specific RT-PCR demonstrated that expression of the
type I receptor gene is a common feature of stromal cells in
vitro (Fig. 2A). This gene
expression is maintained at least up to passage 4 (Fig. 2B). RT-PCR
also demonstrated that these same cultures possess measurable levels of
IGF-IIR messenger RNA (Fig. 3). Specific
stromal binding sites for the IGFs were characterized by displacement
studies. As Fig. 4A demonstrates,
[125I]IGF-I binding to the prostatic stroma could be
competitively inhibited by unlabeled IGF-I and, at 5-fold higher
concentrations, IGF-II. The concentration of insulin required to
produce similar displacement was far in excess of that observed with
either IGF-I or IGF-II. Similarly, using [125I]IGF-II,
the relative affinities for the competing ligands were IGF-II >>
IGF-I > insulin (Fig. 4B). Scatchard analysis proposed a stromal
IGF-I-binding site with an apparent Kd of 4.9 x
10-9 mol/L and a Bmax equivalent to 6.6
x 105 sites/cell. Analysis of the IGF-II displacement data
suggests a binding site with comparable binding affinity
(Kd = 4.7 x 10-9 mol/L); however,
maximal binding was elevated to 3.8 x 106 sites/cell
(Fig. 4B).
|
|
|
Figure 5A shows the effect of 4-day
incubation with IGF-I, at concentrations varying from 0.5–500 ng/mL,
on cell density. At a concentration of 500 ng/mL, IGF-I significantly
(P < 0.05) stimulated cell growth compared to that
under basal conditions, inducing an approximately 80% increase in cell
density over a 96-h period. Likewise, exposure of the same stromal cell
culture to 500 ng/mL IGF-II for 96 h induced a 113% increase in
cell density compared with the control value (Fig. 5B). At 5.0 µg/mL,
the IR3 IGF-IR neutralizing antibody significantly
(P < 0.01) reduced the rate of stromal cell
proliferation even in the presence of stimulatory concentrations of
both IGF-I and IGF-II (Fig. 5, C and D).
|
Camptothecin-induced apoptosis was detected quantitatively in
prostatic stromal cells by DNA fragmentation ELISA. Control cells grown
in SFM/ts exhibited a basal level of apoptosis (Fig. 6A). Addition of camptothecin at 0.1,
1.0, and 5.0 µg/mL induced a significant (P < 0.01)
increase in the apoptotic index above the basal level caused by serum
deprivation. At all concentrations of camptothecin, 500 ng/mL IGF-I
reduced the apoptotic index to control levels (Fig. 6A). As the results
shown in Fig. 6B indicate, specific antagonism of the IGF-IR using
IR3 completely abolished this antiapoptotic activity and, indeed,
initiated cell death to a similar degree as camptothecin even in the
absence of topoisomerase I inhibitor.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The data presented here and in previous reports demonstrate that the prostatic fibromuscular stroma is a rich source of IGF-II and possesses an abundance of receptors for IGF (14, 15). Benign stromal cells grown in SFM, despite exhibiting little or no synthesis of IGF-I, secrete high levels of IGF-II. Furthermore, it is apparent that stromal cells derived from areas of BPH demonstrate elevated transcription of the IGF-II gene compared with cultures obtained from putative normal tissue (14, 15). Coupled with these observations, Dong et al. demonstrated that expression of the IGF type I receptor is also up-regulated in benign stromal cell cultures compared with normal controls, and that both phenomena have their origins in decreased expression of the gene for the transcription factor WT-1. These alterations in the stromal IGF axis have been offered as potential mediators of the abnormal growth in BPH and imply that the IGFs and their associated receptors may represent a potentially novel route for medical intervention.
Our data suggest that IGFs may be a double edged sword for BPH because not only are they mitogenic for prostate tissue, but it is also apparent that they will inhibit apoptosis. Addition of exogenous IGF-I and IGF-II at 500 ng/mL induces almost equivalent increases in the rate of cell proliferation under serum-free conditions. This is the first demonstration that prostatic stromal cells are growth stimulated by IGFs and parallels studies confirming that it is a phenomenon shared by prostatic epithelial cells (27, 28, 29). The measurement of camptothecin-induced increases in the apoptotic index is a crucial observation because it proves that the fibromuscular stroma, previously believed to be strongly resistant to cell death signals (30), can be induced to undergo apoptosis. In addition, it is entirely possible that stromal resistance to apoptosis at least in part is due to high level expression of IGFs, as we have ascertained that, in common with other systems, increases in stromal cell apoptosis arising from exposure to chemotherapeutic agents can be completely inhibited by the addition of IGF-I (31).
RT-PCR detects messenger RNA transcripts for both the type I and type
II IGF receptor in the prostatic stroma in vitro.
Furthermore, competitive binding analyses provide strong evidence for
the presence of distinct IGF-I and IGF-II receptors that differ from
each other in their ability to recognize preferentially either IGF-I or
IGF-II. Scatchard analysis of the IGF-I and IGF-II competition data
proposes an almost identical Kd, whose value is well within
the range of dissociation constants previously reported for the IGF
receptors (1, 28, 32). These binding parameters are, however, not
accompanied by correspondingly similar estimates of maximal binding.
Indeed, it would appear from the data that the putative type II IGF
receptor is more than 5 times as abundant as the IGF-IR. Nonetheless,
despite the apparent prevalence of the IGF-IIR and as has been shown in
previous studies, it is the type I receptor that mediates cellular
responses to the IGFs (5, 6). The IR3 monoclonal antibody has been
observed to potently inhibit the binding of IGF-I to the IGF-IR and to
potently antagonize the growth-promoting and antiapoptotic activities
of the IGFs (16, 28, 33, 34, 35). In prostatic stromal cell cultures,
IR3 significantly inhibits growth stimulation in the presence of
endogenous or exogenous IGFs. In addition, the ability of IGF-I to
block the apoptotic potential of camptothecin is significantly reduced
when access to the IGF-IR is denied. Both of these observations suggest
that the signals so essential to stromal cell well-being cannot be
supplied by a ligand-bound IGF type II receptor.
Clearly, the role of the IGF-IR is fundamentally important to the
fibromuscular stroma in BPH. It is implicated in the etiology of the
disease, demonstrating up-regulation coupled with the ability to
translate the interaction with autocrine-acting ligands into
intracellular mitogenic signals. Furthermore and perhaps more
crucially, it provides the stroma with the machinery to resist
apoptosis. Indeed, even in the absence of external apoptotic stimuli
and regardless of the activation of other growth factor receptors, the
loss of IGF-IR signaling through IR3 binding is sufficient to
trigger stromal cell death. All of these findings lend support to the
use of therapies based on the inhibition of normal IGF-IR function as
an alternative or supplement to standard steroid-based therapies for
BPH.
Received April 23, 1998.
Revised June 3, 1998.
Accepted June 15, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IR-3 inhibits non-small cell
lung cancer growth in vitro and in vivo. J
Cell Biochem. 24(Suppl):269–275.
This article has been cited by other articles:
 |
|
 |
|
  G. P. Finkielstain, P. Forcinito, J. C. K. Lui, K. M. Barnes, R. Marino, S. Makaroun, V. Nguyen, J. E. Lazarus, O. Nilsson, and J. Baron An Extensive Genetic Program Occurring during Postnatal Growth in Multiple Tissues Endocrinology, April 1, 2009; 150(4): 1791 - 1800. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Kleinberg, W. Ruan, D. Yee, K. T. Kovacs, and S. Vidal Insulin-Like Growth Factor (IGF)-I Controls Prostate Fibromuscular Development: IGF-I Inhibition Prevents Both Fibromuscular and Glandular Development in Eugonadal Mice Endocrinology, March 1, 2007; 148(3): 1080 - 1088. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. Jeschke, U. Bolder, D. H. Chung, R. Przkora, U. Mueller, J. C. Thompson, S. E. Wolf, and D. N. Herndon Gut Mucosal Homeostasis and Cellular Mediators after Severe Thermal Trauma and the Effect of Insulin-Like Growth Factor-I in Combination with Insulin-Like Growth Factor Binding Protein-3 Endocrinology, January 1, 2007; 148(1): 354 - 362. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Colao, D. Ferone, P. Marzullo, and G. Lombardi Systemic Complications of Acromegaly: Epidemiology, Pathogenesis, and Management Endocr. Rev., February 1, 2004; 25(1): 102 - 152. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. O. Roberts, D. J. Jacobson, C. J. Girman, T. Rhodes, G. G. Klee, M. M. Lieber, and S. J. Jacobsen Insulin-like Growth Factor I, Insulin-like Growth Factor Binding Protein 3, and Urologic Measures of Benign Prostatic Hyperplasia Am. J. Epidemiol., May 1, 2003; 157(9): 784 - 791. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. N. N. Han, M. Post, A. K. Tanswell, and S. J. Lye Insulin-Like Growth Factor-I Receptor-Mediated Vasculogenesis/Angiogenesis in Human Lung Development Am. J. Respir. Cell Mol. Biol., February 1, 2003; 28(2): 159 - 169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Monti, F. Di Silverio, R. Iraci, C. Martini, S. Lanzara, P. Falasca, M. Poggi, A. Stigliano, F. Sciarra, and V. Toscano Regional Variations of Insulin-Like Growth Factor I (IGF-I), IGF-II, and Receptor Type 1 in Benign Prostatic Hyperplasia Tissue and Their Correlation with Intraprostatic Androgens J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1700 - 1706. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
